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, 95 (23), 13465-70

The Reactive Site Loop of the Serpin SCCA1 Is Essential for Cysteine Proteinase Inhibition

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The Reactive Site Loop of the Serpin SCCA1 Is Essential for Cysteine Proteinase Inhibition

C Schick et al. Proc Natl Acad Sci U S A.

Abstract

The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin-serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.

Figures

Figure 1
Figure 1
Kinetic analysis of SCCA1 hinge mutants. The effect of hinge mutations on the inhibition of catS was measured under pseudo-first-order conditions by using the progress-curve method. Wild-type SCCA1 (□), SCCA1 AlaP14Thr mutant (■), or SCCA1 AlaP14Arg mutant (•) were incubated with catS and substrate at 25°C in cathepsin buffer, pH 5.5. The progress of catS inactivation was followed by measuring the relative fluorescence (RFU) over time. The rate of inhibition of catS by the AlaP14Thr SCCA1 mutant was kass = 3.9 × 105 M−1⋅s−1. CatS without inhibitor (○) served as a control. A set of curves from a representative experiment is depicted. Concentrations of reagents: inhibitor, 250 nM; enzyme, 10 nM; substrate [(Z-FR)2-R110], 5 μM.
Figure 2
Figure 2
SDS/PAGE analysis of the interaction of SCCA1 mutants and different proteinases. SCCA1 or SCCA1 mutants were incubated with proteinases at 25°C for 15 min in reaction buffer and then heated at 95°C for 5 min in gel-loading buffer. Protein mixtures were separated by SDS/PAGE and stained with Coomassie blue. Positions of the molecular weight markers are noted to the left of the gel. The molecular mass of SCCA1 and SCCA1 mutants was ≈71 kDa; catS, ≈24 kDa; chymotrypsin, ≈25 kDa; α1-proteinase inhibitor, ≈52 kDa; and trypsin, ≈22 kDa. (A) The interaction of the AlaP14Arg SCCA1 mutant with catS. Lanes: 1, SCCA1 (1.25 μg); 2, SCCA1 with catS (0.3 μg); 3, SCCA1 AlaP14Arg (1.4 μg); 4, SCCA1 AlaP14Arg with catS. The positions of the native (arrow) and cleaved (arrowhead) inhibitors are indicated. (B) The interaction of the ThrP3′Ala SCCA1 mutant with chymotrypsin. Lanes: 1, SCCA1 (5.0 μg); 2, SCCA1 with chymotrypsin (1.0 μg); 3, SCCA1 ThrP3′Ala (5.0 μg); 4, SCCA1 ThrP3′Ala with chymotrypsin; 5, α1-proteinase inhibitor (2.7 μg); 6, α1-proteinase inhibitor with chymotrypsin serve as controls for chymotrypsin activity. The positions of the inhibitors (arrows), α1-proteinase inhibitor–chymotrypsin complex (open arrowhead), and the wild-type and mutant SCCA1 degradation products (closed arrowheads) are indicated. (C) Formation of SDS–stable complexes of trypsin and SCCA1 GlyP2Arg. Lanes: 1, SCCA1 (5.0 μg); 2, SCCA1 with trypsin (1.0 μg); 3, SCCA1 GlyP2Arg (6.0 μg); 4, SCCA1 GlyP2Arg and trypsin. The positions of the inhibitors (arrow), SCCA1 GlyP2Arg-trypsin complex (arrowhead), and the wild-type and mutant degradation products (closed arrowheads) are indicated.
Figure 3
Figure 3
Kinetic analysis of RSL variable-region mutants. The effect of mutations in the variable region of the RSL on catS inhibition was measured under pseudo-first-order conditions by using the progress-curve method, as described in Fig. 1. Sets of curves from representative experiments are depicted. (A) Loop swap mutants. The rates of product formation by catS in the presence of wild-type SCCA2 (□), the SCCA1(2) mutant (▴) (containing the SCCA2 RSL), wild-type SCCA1 (•), and SCCA2(1) mutant (■) (containing the SCCA1 RSL). CatS without inhibitor (○) served as a control. The rate of catS inhibition by SCCA2(1) was kass = 1.2 × 105 M−1⋅s−1. Concentrations of reagents: inhibitor, 250 nM; enzyme, 10 nM; substrate [(Z-FR)2-R110], 5 μM. (B) P1 mutants. The rate of inhibition of catS by the GlyP2Arg SCCA1 mutant (■) (kass = 1.8 × 104 M−1⋅s−1) and the GlyP2Glu SCCA1 mutant (□) (kass = 1.1 × 105 M−1⋅s−1) was compared with that of wild-type SCCA1 (○). The curve of the GlyP2Glu SCCA1 mutant is obscured by that of the wild-type SCCA1. CatS without inhibitor (•) served as a control. Concentrations of reagents: inhibitor, 360 nM; enzyme, 13.5 nM; substrate [(Z-FR)2-R110], 10 μM. (C) P2 mutants. The rate of inhibition of catS by the PheP3Ala SCCA1 mutant (■) and the PheP3Leu SCCA1 mu tant (•) (kass = 3.3 × 105 M−1⋅s−1) was compared with that of wild-type SCCA1 (□). CatS without inhibitor (○) served as a control. Concentrations of reagents: inhibitor, 250 nM; enzyme, 10 nM; substrate [(Z-FR)2-R110], 5 μM.
Figure 4
Figure 4
Kinetic and biochemical analysis of the interaction of trypsin and SCCA1 GlyP2Arg mutant. (A) Kinetic analysis of trypsin inhibition by SCCA1 GlyP2Arg using the progress-curve method. Trypsin (7.7 nM) alone (○) or with SCCA1 (□, 509 nM) or SCCA1 GlyP2Arg (•, 517 nM) was incubated with substrate (EGR-pNA, 0.8 mM) at 25°C in PBS reaction buffer under pseudo-first-order conditions. The progress of trypsin inactivation was followed by measuring the ΔA405 for the reaction over time. The SCCA1 GlyP2Arg mutant inhibited trypsin with a second-order rate constant of kass = 3.9 × 104 M−1⋅s−1. A set of curves from a representative experiment is depicted. (B) Stoichiometry of inhibition of trypsin by SCCA1 GlyP2Arg. Trypsin (4 μM) was incubated with different concentrations of SCCA1 GlyP2Arg (0–4 μM) at 25°C for 30 min in PBS. Residual trypsin activity was measured by adding the substrate (EGR-pNA) and measuring the ΔA405. Fractional activity was the ratio of the velocity of inhibited enzyme (vi) to the velocity of uninhibited control (v0). The stoichiometry of inhibition was determined by using linear regression to extrapolate the inhibitor and enzyme ratio, resulting in complete inhibition. (C) MALDI analysis of trypsin and SCCA1 GlyP2Arg cleavage products. Trypsin (2.2 μg) was incubated with SCCA1 GlyP2Arg (10 μg) for 5 min at 25°C. The reaction mixture then was separated by MALDI MS.

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